Complex of gadolinium and a chelating ligand derived from a diastereoisomerically enriched pcta and preparation and purification process

ABSTRACT

The present invention relates to a complex of formula (II) constituted of at least 90% of a diastereoisomeric excess comprising a mixture of isomers II-RRR and II-SSS of formulae: 
     
       
         
         
             
             
         
       
     
     The present invention also relates to a process for preparing and purifying said complex of formula (II), and also to a composition comprising said complex.

The present invention relates to a novel process for preparing andpurifying a complex of gadolinium and of a PCTA-based chelating ligand,which makes it possible to obtain preferentially stereoisomers of saidcomplex which have physicochemical properties that are most particularlyadvantageous for applications as contrast agent in the field of medicalimaging, notably for magnetic resonance imaging. The present inventionalso relates to the diastereoisomerically enriched complex per se, to acomposition comprising said complex, and also to a process for preparingthe corresponding chelating ligand by decomplexation of said complex,and to the ligand per se.

Many contrast agents based on chelates of lanthanides (paramagneticmetal), in particular gadolinium (Gd), are known, for example describedin U.S. Pat. No. 4,647,447. These products are often grouped under theterm GBCA (gadolinium-based contrast agent). Several products aremarketed, among which are macrocyclic chelates such as megluminegadoterate based on DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid),gadobutrol based on DO3A-butrol, gadoteridol based on HPDO3A, and alsolinear chelates, notably based on DTPA (diethylenetriaminepentaaceticacid) or on DTPA-BMA (gadodiamide ligand).

Other products, some of which are under development, represent a newgeneration of GBCA. They are essentially complexes of macrocyclicchelates, such as bicyclopolyazamacrocyclocarboxylic acid (EP 0 438 206)or PCTA derivatives (i.e. derivatives comprising a minima the3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triaceticacid chemical structure), as described in EP 1 931 673.

The complexes of PCTA-based chelating ligands described in EP 1 931 673notably have the advantage of being relatively easy to synthesizechemically and, in addition, of having relaxivity superior to that ofthe other GBCAs (relaxivity r1 which may be up to 11-12 mM⁻¹.s⁻¹ inwater) currently on the market, this relaxivity corresponding to theefficiency of these products and thus to their contrasting power.

In the body, chelates (or complexes) of lanthanide—and notably ofgadolinium—are in a state of chemical equilibrium (characterized by itsthermodynamic constant K_(therm)), which may lead to an undesiredrelease of said lanthanide (see equation 1 below):

Ch+Ln

  (equation 1)

Complexation chemical equilibrium between the chelate or ligand (Ch) andthe lanthanide (L_(n)) to give the complex Ch-L_(n).

Since 2006, a pathology known as NSF (Nephrogenic Systemic Fibrosis orfibrogenic dermopathy), has been at least partly linked to the releaseof free gadolinium into the body. This disease has alerted healthauthorities with regard to gadolinium-based contrast agents marketed forcertain categories of patients.

Strategies were thus put into place to solve in an entirely safe mannerthe complex problem of patient tolerance and to limit, or eveneliminate, the risk of undesired lanthanide release afteradministration. This problem is all the more difficult to solve sincethe administration of contrast agents is often repeated, whether duringdiagnostic examinations or for the adjustment of doses and themonitoring of the efficacy of a therapeutic treatment.

In addition, mention has been made since 2014 of a possible cerebraldeposition of gadolinium after repeated administrations ofgadolinium-based products, more particularly of linear gadoliniumchelates, such a deposition having been sparingly or not at all reportedwith gadolinium macrocyclic chelates, such as Dotarem®. Consequently,various countries have decided either to withdraw the majority of thelinear chelates from the market, or to drastically limit theirindications for use, given their stability which is deemed insufficient.

A first strategy for limiting the risk of lanthanide release into thebody thus consists in opting for complexes which are distinguished bythermodynamic and/or kinetic stabilities that are as high as possible.The reason for this is that the more stable the complex, the more theamount of lanthanide released over time will be limited.

Other approaches for improving the tolerance of chelates of lanthanide(notably of gadolinium) are described in the prior art. U.S. Pat. No.5,876,695, which is more than 30 years old, reports, for example,formulations comprising, besides the lanthanide chelate, an additionalcomplexing agent, intended for preventing undesired in-vivo release ofthe lanthanide, by complexing the leached lanthanide (Gd³⁺ metal ion).The additional chelating agent may be introduced into the formulationeither in its free form, or in the form of a weak complex, typically ofcalcium, sodium, zinc or magnesium. While it may, possibly, be distinctfrom the constituent ligand of the active complex, it is neverthelessimportant for the complex it forms with the released lanthanide to beless stable than the active complex, so as to prevent a trans-ligationreaction between the active complex and the additional chelate, whichwould notably have the effect of totally consuming said additionalligand, which could then no longer trap the leached lanthanide. Thisrisk of consumption of the additional chelating agent by trans-ligationis more pronounced when it is added in free form than in the form of acalcium complex, for example.

Thus, in the two strategies described above, it is important for theactive complex to be as stable as possible.

However, the complexes of PCTA-based chelating ligands comprising astructure of pyclene type described in EP 1 931 673, while having goodkinetic stability, generally have a thermodynamic constant which islower than that of complexes of the other cyclene-based macrocycles.

This is notably the case for the complex of formula (II) representedbelow:

Indeed, as is notably described in WO 2014/174120, the thermodynamicequilibrium constant corresponding to the reaction for the formation ofthe complex of formula (II), also known as the stability constant, is10^(14.9) (i.e. log (K_(therm))=14.9). For comparative purposes, thestability constant of the gadolinium complex of1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA-Gd) is10^(25.6) (i.e. log (K_(therm))=25.6).

It should be noted, however, that the complex of formula (II)corresponds to several stereoisomers, notably due to the presence of thethree asymmetric carbon atoms located in the α position on the sidechains of the complex, relative to the nitrogen atoms of the macrocycleonto which said side chains are grafted. These three asymmetric carbonsare marked with an asterisk (*) in formula (II) represented above.

Thus, the synthesis of the complex of formula (II) as described in EP 1931 673 results in the production of a mixture of stereoisomers.

The aminopropanediol groups of the side chains of the complex of formula(II) also include an asymmetric carbon. Thus, the complex of formula(II) comprises in total six asymmetric carbons, and thus exists in theform of 64 configurational stereoisomers. However, in the rest of thedescription, the only source of stereoisomerism considered for a givenside chain will, for the sake of simplicity, be that corresponding tothe asymmetric carbon bearing the carboxylate group, marked with anasterisk (*) in formula (II) represented above.

Since each of these three asymmetric carbons may be of R or S absoluteconfiguration, the complex of formula (II) exists in the form of eightfamilies of stereoisomers, referred to hereinbelow as II-RRR, II-SSS,II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS. More precisely,according to the usual nomenclature in stereochemistry, the complex offormula (II) exists in the form of eight families of diastereoisomers.

The use of the term “family” is justified in that each of these familiesincludes several stereoisomers, notably due to the presence of anasymmetric carbon within the aminopropanediol group, as mentionedpreviously.

Nevertheless, since, in the rest of the description, the stereoisomerismassociated with the asymmetric carbon of a given aminopropanediol groupwill not be considered, the terms isomers, stereoisomers ordiastereoisomers II-RRR, II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSRand II-SRS will be used without distinction, without stating that eachcorresponds to a family of stereoisomers.

The inventors have succeeded in separating and in identifying byhigh-performance liquid chromatography (HPLC) and byultra-high-performance liquid chromatography (UHPLC) four unresolvedpeaks or groups of isomers of the complex of formula (II) obtainedaccording to the process of the prior art, corresponding to fourdifferent elution peaks characterized by their retention time on thechromatogram, which will be referred to in the rest of the descriptionas iso1, iso2, iso3 and iso4. By performing the process described in EP1 931 673, the respective contents of the groups iso1, iso2, iso3 andiso4 in the mixture obtained are as follows: 20%, 20%, 40% and 20%.

They then discovered that these various groups of isomers had differentphysicochemical properties, and determined that the group of isomersknown as iso4, which comprises a mixture of the isomers II-RRR andII-SSS of formulae (II-RRR) and (II-SSS) represented below, proves to bethe most advantageous as contrast agent for medical imaging.

Indeed, iso4 is distinguished, surprisingly, by a thermodynamicstability that is markedly superior to that of the mixture ofdiastereoisomers in the form of which the complex of formula (II) isobtained by performing the process described in EP 1 931 673.Specifically, its equilibrium thermodynamic constant K_(therm iso4) isequal to 10^(18.7) (i.e. log (K_(therm iso4))=18.7) this value havingbeen determined by performing the method in Pierrard et al., ContrastMedia Mol. Imaging, 2008, 3, 243-252 and Moreau et al., Dalton Trans.,2007, 1611-1620.

Besides, iso4 is the group of isomers which has the best kinetic inertia(also known as kinetic stability) among the four groups isolated by theinventors. Specifically, the inventors evaluated the kinetic inertia ofthe four groups of isomers by studying their decomplexation kinetics inacidic aqueous solution (pH=1.2), at 37° C. The half-life time values(T_(1/2)) which were determined for each of the groups of isomers areindicated in table 1 below, the half-life time corresponding to the timeafter which 50% of the amount of complex initially present has beendissociated, according to the following decomplexation reaction(equation 2):

TABLE 1 decomplexation kinetics for the groups of isomers iso1 to iso4Groups of isomers T_(1/2) (pH 1.2-37° C.) Iso1 18 hours Iso2 6 hoursIso3 8 days Iso4 27 days

For comparative purposes, gadobutrol or gadoterate, which aremacrocyclic gadolinium complexes, respectively have a kinetic inertia of18 hours and of 4 days under the same conditions, whereas lineargadolinium complexes such as gadodiamide or gadopentetate dissociateinstantaneously.

In addition, iso4 is chemically more stable than iso3, notably. Thereason for this is that the amide functions of the complex of formula(II) are liable to be hydrolysed. The hydrolysis reaction of an amidefunction (equation 3) results in the formation of a dicoupled impurity,which is accompanied by the release of 3-amino-1,2-propanediol. Theinventors studied the kinetics of the hydrolysis reaction of the complexof formula (II) in aqueous solution at pH 13 and observed that the amidefunctions of iso4 are more stable with respect to hydrolysis than thoseof iso3.

As regards the relaxivity of the various groups of isomers, i.e. theirefficiency as contrast agent, the measurements taken demonstrate acontrasting power that is relatively equivalent for the groups iso1,iso2 and iso4, and reduced efficiency for iso3 (see table 2).

TABLE 2 relaxivity of the groups of isomers iso1 to iso4 at 37° C.Groups of r1 20 MHz r1 60 MHz isomers (mM⁻¹ · s⁻¹) (mM⁻¹ · s⁻¹) Iso112.6 12.5 Iso2 13.3 12.9 Iso3  8.0  8.1 Iso4 12.9 13.0

The inventors have succeeded in developing a novel process for preparingand purifying the complex of formula (II), making it possible to obtainpreferentially the diastereoisomers II-RRR and II-SSS of said complex,which have particularly advantageous physicochemical properties. Theprocess according to the invention comprises a step of isomericenrichment, by conversion of the least stable stereoisomers into themost stable stereoisomers, which, surprisingly, while being performed onthe hexaacid intermediate complex and not on the final complex, makes itpossible to obtain very predominantly the most stable isomers of thecomplex of formula (II).

The implementation of a process which makes it possible to obtainpredominantly the diastereoisomers of interest is unquestionablyadvantageous when compared with the alternative consisting in preparingthe mixture of stereoisomers, then subsequently attempting to separatethe diastereoisomers according to the usual techniques and thus toisolate the isomers of interest using any separation technique that iswell known in the art. Indeed, besides the fact that it is easier toperform a process not involving a step of separation of diastereoisomerson an industrial scale, the absence of separation firstly affordsconsiderable time-saving and secondly makes it possible to improve theoverall yield of the process, by limiting as much as possible theproduction of the undesired diastereoisomers which would ultimately bediscarded. Moreover, the usual separation techniques generally involvean abundant use of solvents, which, beyond the financial cost, is notdesirable for environmental reasons. Furthermore, chromatography onsilica is in particular to be avoided, given the health risks inherentin professional exposure to silica, which is classified as carcinogenicto humans (group 1) by the International Agency for Research on Cancer.

As indicated previously, the process for preparing the complex offormula (II) developed by the inventors is based on a step of isomericenrichment of the intermediate hexaacid gadolinium complex of formula(I) represented below:

The complex of formula (I) corresponds to several stereoisomers, due tothe presence of the three asymmetric carbon atoms located in the αposition on the side chains of the complex, relative to the nitrogenatoms of the macrocycle onto which said side chains are grafted. Thesethree asymmetric carbons are marked with an asterisk (*) in formula (I)represented above.

Since each of the three asymmetric carbons bearing a carboxylatefunction may be of R or S absolute configuration, the complex of formula(I) exists in the form of eight stereoisomers, referred to hereinbelowas I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR and I-SRS. Moreprecisely, according to the usual nomenclature in stereochemistry, thecomplex of formula (I) exists in the form of four pairs of enantiomers,which are mutual diastereoisomers.

The inventors have succeeded in separating and in identifying byhigh-performance liquid chromatography (HPLC) and byultra-high-performance liquid chromatography (UHPLC) four unresolvedpeaks or groups of isomers of the complex of formula (I) obtainedaccording to the process described in EP 1 931 673, corresponding tofour different elution peaks characterized by their retention time onthe chromatogram, which will be referred to in the rest of thedescription as isoA, isoB, isoC and isoD.

IsoD crystallizes from water. X-ray diffraction analysis enabled theinventors to determine the crystal structure of this group of isomers,and thus to discover that it comprises the diastereoisomers I-RRR andI-SSS of the complex of formula (I), of formulae (I-RRR) and (I-SSS)represented below.

It should be noted that the diastereoisomers I-RRR and I-SSS of thecomplex of formula (I) are enantiomers of each other.

The isomeric enrichment step of the process of the invention aims atenriching the intermediate hexaacid gadolinium complex of formula (I) inisoD.

The synthesis of the complex of formula (II) notably involves conversionof the carboxylic acid functions of the intermediate hexaacid complex offormula (I) into amide functions. This amidation reaction does notmodify the absolute configuration of the three asymmetric carbon atomsof the complex of formula (I).

Thus, when the amidation reaction is performed on the hexaacid complexof formula (I) enriched in isoD obtained previously, it makes itpossible to obtain the complex of formula (II) enriched in iso4.

Moreover, the purification process developed by the inventors makes itpossible, when it is performed following the process for preparing thecomplex of the abovementioned formula (II), to obtain the complex offormula (II) with an optimized isomeric profile, but also a markedlyimproved impurity profile.

This diastereoisomerically enriched and purified complex of improvedstability can consequently be formulated with a free macrocyclic ligand,such as free DOTA, instead of a calcium complex of DOTA, the use ofwhich was recommended in WO 2014/174120. The use of free DOTA notablyhas an advantage from an industrial viewpoint, in the sense that itmakes it possible to eliminate a step of the process for synthesizingthe formulation as described in WO 2014/174120, namely the addition ofCaCl₂.

Complex of Formula (II)

The present invention thus relates firstly to a complex of formula (II):

constituted of at least 80% of a diastereoisomeric excess comprising amixture of isomers II-RRR and II-SSS of formulae:

In the context of the present invention, the term “diastereoisomericexcess” is intended to denote, as regards the complex of formula (II),the fact that said complex is predominantly present in the form of anisomer or group of isomers chosen from the diastereoisomers II-RRR,II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS. Saiddiastereoisomeric excess is expressed as a percentage and corresponds tothe amount represented by the predominant isomer or group of isomersrelative to the total amount of the complex of formula (II). It isunderstood that this percentage may be on either a molar or mass basis,since isomers have, by definition, the same molar mass.

In one particular embodiment, the complex of formula (II) according tothe invention has at least 85%, notably at least 90%, in particular atleast 92%, preferably at least 94%, advantageously at least 97%, moreadvantageously at least 99% of the diastereoisomeric excess comprisingthe mixture of isomers II-RRR and II-SSS.

Preferably, said diastereoisomeric excess is constituted of at least70%, notably of at least 80%, advantageously of at least 90%, preferablyof at least 95% of the mixture of isomers II-RRR and II-SSS.

Advantageously, said diastereoisomeric excess consists of the mixture ofisomers II-RRR and II-SSS.

The term “mixture of isomers II-RRR and II-SSS” also covers, byextension, the case where only one of the isomers, whether it be II-RRRor II-SSS, is present. However, the term “mixture of isomers II-RRR andII-SSS” preferentially denotes all the cases in which each of theisomers II-RRR and II-SSS is present in a variable but non-zero amount.

In a preferred embodiment, the isomers II-RRR and II-SSS are present insaid mixture in a ratio of between 65/35 and 35/65, notably between60/40 and 40/60, in particular between 55/45 and 45/55. Advantageously,the isomers II-RRR and II-SSS are present in the mixture in a 50/50ratio.

More particularly, the diastereoisomeric excess as defined previouslycorresponds to peak 4 in the UHPLC plot (i.e. the fourth unresolved peakof isomers in the order of elution and corresponding to iso4),characterized by a retention time of between 6.0 and 6.6 minutes,typically of about 6.3 minutes, said plot being obtained using the UHPLCmethod described below.

For the purposes of the present invention, the term “UHPLC plot” meansthe profile of the concentrations measured by the detector after passageand separation of a mixture of compounds (in this instance of isomers ofa compound) on a stationary phase as a function of time for a givencomposition and a given flow rate of eluent. The UHPLC plot isconstituted of various peaks or unresolved peaks characteristic of thecompound or of the mixture of compounds analysed.

UHPLC method:

-   -   Waters Cortecs® UPLC T3 150×2.1 mm−1.6 μm column.    -   It is a reverse-phase UPLC column with spherical particles        constituted of a core, which is preferentially very hard, made        of silica surrounded by a porous silica with trifunctional C18        (octadecyl) grafting, and the silanols of which have been        treated with capping agents (end-capped). It is also        characterized by a length of 150 mm, an inside diameter of 2.1        mm, a particle size of 1.6 μm, a porosity of 120 A and a carbon        content of 4.7%.    -   Preferentially, the stationary phase used should be compatible        with the aqueous mobile phases.    -   analytical conditions:

Aqueous solution of the complex Sample of formula (II) at 2.0 mg/mLColumn temperature 40° C. Sample temperature Room temperature (20-25°C.) Flow rate 0.3 mL/min Injection volume 1 μL UV detection 200 nm

-   -   mobile phase gradient (% v/v):

Time acetonitrile H₂SO₄ (min) (100%) (aqueous solution at 0.0005% v/v) 0  1 99  3  5 95 12 10 90

Composition Comprising the Complex of Formula (II)

The present invention relates secondly to a composition comprising:

-   -   the complex of formula (II) constituted of at least 80% of a        diastereoisomeric excess comprising a mixture of isomers II-RRR        and II-SSS and    -   a free macrocyclic ligand.

In the present description, the terms “macrocyclic ligand” or“macrocyclic chelate” may be used without distinction.

In the context of the present invention, the term “macrocycle” denotes aring typically including at least nine atoms, whether they are carbonatoms or heteroatoms, and the term “macrocyclic ligand” or “macrocyclicchelate” is a polydentate, at least bidentate, ligand.

For the purposes of the present invention, the term “free macrocyclicligand” means the macrocyclic ligand in free form, i.e. not complexed,in particular with metals—including lanthanides and actinides—or withalkaline-earth metal cations such as calcium or magnesium. Inparticular, the free macrocyclic ligand is not in the form of a complexwith gadolinium, and is not introduced into the composition in the formof a weak complex, typically of calcium, sodium, zinc or magnesium, asdescribed in U.S. Pat. No. 5,876,695, the presence of said cations intrace amount in the composition and thus of the corresponding complexesnot, however, being excluded.

As discussed previously, the formulation of the complex of formula (II)with a free macrocyclic ligand, and not a weak complex of saidmacrocyclic ligand as recommended in EP 1 931 673, is made possible bythe improved stability of the diastereoisomerically enriched complex offormula (II) according to the invention.

In a preferred embodiment, the complex of formula (II) present in thecomposition of the invention has at least 85%, notably at least 90%, inparticular at least 92%, more particularly at least 94%, preferably atleast 97%, advantageously at least 99% of the diastereoisomeric excesscomprising the mixture of isomers II-RRR and II-SSS.

Preferably, said diastereoisomeric excess is constituted of at least70%, notably of at least 80%, advantageously of at least 90%, preferablyof at least 95% of the mixture of isomers II-RRR and II-SSS.

Advantageously, said diastereoisomeric excess consists of the mixture ofisomers II-RRR and II-SSS.

The term “mixture of isomers II-RRR and II-SSS” also covers, byextension, the case where only one of the isomers, whether it be II-RRRor II-SSS, is present. However, the term “mixture of isomers II-RRR andII-SSS” preferentially denotes all the cases in which each of theisomers II-RRR and II-SSS is present in a variable but non-zero amount.

In a preferred embodiment, the isomers II-RRR and II-SSS are present insaid mixture in a ratio of between 65/35 and 35/65, notably between60/40 and 40/60, in particular between 55/45 and 45/55. Advantageously,the isomers II-RRR and II-SSS are present in the mixture in a 50/50ratio.

In one advantageous embodiment, the composition according to theinvention has a concentration of free gadolinium of less than 1 ppm(m/v), preferentially less than 0.5 ppm (m/v).

In the present description, unless otherwise mentioned, the terms “Gd”,“gadolinium” and “Gd³⁺” are used without distinction to denote the Gd³⁺ion. By extension, it may also be a source of free gadolinium, such asgadolinium chloride (GdCl₃) or gadolinium oxide (Gd₂O₃).

In the present invention, the term “free Gd” denotes the non-complexedforms of gadolinium, which are preferably available for complexation. Itis typically the Gd³⁺ ion dissolved in water. By extension, it may alsobe a source of free gadolinium, such as gadolinium chloride (GdCl₃) orgadolinium oxide (Gd₂O₃).

Gadolinium in free form is typically measured by colorimetric assay,generally xylenol orange or Arsenazo (III). In the absence of a metalion (such as gadolinium), these indicators have a specific colour: atacidic pH, xylenol orange is yellow, whereas Arsenazo is pink. In thepresence of gadolinium, their colour changes to violet.

Visual determination of the colour change of the solution makes itpossible to verify the presence or absence of gadolinium in thesolution.

Moreover, it is possible to quantitatively measure the free gadoliniumthat is in the solution via a back titration, for example using EDTA as“weak” gadolinium chelate. In such an assay, the coloured indicator isadded until a violet colour is obtained. EDTA, a gadolinium ligand, isthen added dropwise to the mixture. Since EDTA is a stronger complexingagent than the coloured indicator, the gadolinium changes ligand andleaves the coloured indicator to become preferentially complexed withEDTA. The coloured indicator thus gradually regains its non-complexedform.

When the amount of EDTA added is equal to the initial amount of free Gd,the coloured indicator is entirely in its free form and the solution“turns” yellow. Since the amount of EDTA added is known, this makes itpossible to know the initial amount of free Gd in the solution to beassayed.

These methods are well known to those skilled in the art and are notablydescribed in Barge et al. (Contrast Media and Molecular Imaging 1, 2006,184-188).

These colorimetric methods are thus usually performed on a solutionwhose pH is between 4 and 8. The reason for this is that outside thesepH ranges, the accuracy of the measurement may be affected due to amodification (or even suppression) of the colour change.

Thus, if need be, the pH of the sample to be assayed is adjusted to bebetween 4 and 8. Notably, if the pH of the sample is acidic, and inparticular less than 4, the pH is advantageously adjusted by adding abase, and the measurement of the free Gd is then performed on the sampleat the adjusted pH.

The composition according to the invention thus has stability over time,i.e. its composition remains in accordance with the specifications interms of concentration of free gadolinium (in particular itsconcentration of free Gd remains less than 1 ppm (m/v)), over a periodof at least 3 years, preferentially of at least 4 years or morepreferentially of at least 5 years, notably in terms of content of freeparamagnetic metal. According to the ICH guidelines, observation of thisstability for six months at 40° C. is considered as a good indication ofstability for 3 years at 25° C.

In one particular embodiment, the composition according to the inventionhas a concentration of between 0.01 and 1.5 mol.L⁻¹, preferentiallybetween 0.2 and 0.7 mol.L⁻¹, more preferentially between 0.3 and 0.6mol.L⁻¹ of complex of formula (II) described above.

The complex of formula (II) is assayed via the methods known to thoseskilled in the art. It may notably be assayed after mineralization andassay of the total gadolinium present in the composition, by atomicemission spectrometry (also known as ICP-AES or ICP Atomic EmissionSpectrometry).

The content of complex of formula (II) allows this composition to havean optimum contrasting power while at the same time having asatisfactory viscosity. Specifically, below 0.01 mol.L⁻¹ of complex offormula (II) described above, the performance qualities as a contrastproduct are less satisfactory, and at a concentration above 1.5 mol.L⁻¹,the viscosity of this composition becomes too great for easy handling.

In one particular embodiment, the composition according to the inventioncomprises between 0.002 and 0.4 mol/mol %, notably between 0.01 and 0.3mol/mol %, preferably between 0.02 and 0.2 mol/mol % and morepreferentially between 0.05 and 0.15 mol/mol % of free macrocyclicligand relative to the complex of formula (II).

Advantageously, the macrocyclic ligand is selected from the groupconstituted of DOTA, NOTA, DO3A, BT-DO3A, HP-DO3A, PCTA, DOTA-GA andderivatives thereof.

Preferably, it is DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

The concentration of free DOTA in the composition is typically measuredby back titration with copper, for example using copper sulfate assource of copper ions.

In this method, which is well known to those skilled in the art, asolution containing a known initial concentration Q_(o) of coppersulfate is preferentially used, this concentration being greater thanthe amount of free ligand in the solution. The solution to be assayed,containing free DOTA in an amount Q₁ to be determined, is added to thiscopper sulfate solution. DOTA is a very good copper complexing agent:the formation of a DOTA-copper complex is thus observed.

Back titration of the copper remaining free in the solution is thenadvantageously performed by potentiometry. To do this, EDTA is, forexample, added dropwise to the mixture. The EDTA complexes the freecopper in solution without, however, decomplexing the DOTA-copper, sinceDOTA is a stronger complexing agent than EDTA. When the amount of EDTAadded Q₂ is equal to the amount of free copper in solution, a suddendrop in the potential of the solution is observed.

Knowing the initial amount of copper Q₀ and the amount of EDTA added Q₂,subtraction of these two values Q₀−Q₂ gives the amount of free DOTA inthe solution to be assayed Q₁.

Alternatively, HPLC methods may be used, notably the HILIC LC-UV method.

These measuring methods (in particular the potentiometric methods) areperformed on solutions whose pH is advantageously between 4 and 8. Thus,if need be, the pH of the sample to be assayed is adjusted to be between4 and 8. Notably, if the pH of the sample is acidic, and in particularless than 4, the pH is advantageously adjusted by adding a base such asmeglumine, and the measurement of the free DOTA is then performed on thesample at the adjusted pH.

Preferentially, the proportions specified in the present invention andin particular above are proportions before sterilization of thecomposition.

Advantageously, the pH of the composition is between 4.5 and 8.5,preferentially between 5 and 8, advantageously between 6 and 8, notablybetween 6.5 and 8. These pH ranges notably make it possible to limit theappearance of certain impurities and to promote the complexation of theparamagnetic metal ion M.

In particular, the composition according to the invention may bebuffered, i.e. it may also comprise a buffer chosen from common buffersestablished for the pH range 5 to 8, preferentially among lactate,tartrate, malate, maleate, succinate, ascorbate, carbonate, Tris(Tris(hydroxymethyl)aminomethane), HEPES(2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid) and MES(2-morpholinoethanesulfonic acid) buffers and mixtures thereof, andpreferentially a buffer chosen from Tris, lactate, tartrate, carbonateand MES buffers and mixtures thereof. Advantageously, the compositionaccording to the invention comprises the Tris buffer.

The composition that is the subject of the invention is preferentiallysterile.

Process for Preparing the Complex of Formula (II)

The present invention also relates to a process for preparing thecomplex of formula (II), comprising the following successive steps:

-   -   a) Complexation of the hexaacid of formula (III) below:

-   -   with gadolinium to obtain the hexaacid gadolinium complex of        formula (I) as defined previously,    -   b) Isomerization by heating the hexaacid gadolinium complex of        formula (I) in an aqueous solution at a pH of between 2 and 4,        to obtain a diastereoisomerically enriched complex constituted        of at least 80% of a diastereoisomeric excess comprising a        mixture of the isomers I-RRR and I-SSS of said hexaacid        gadolinium complex of formula (I), and    -   c) Formation, starting with the diastereoisomerically enriched        complex obtained in step b), of the complex of formula (II), by        reaction with 3-amino-1,2-propanediol.

In the present description, unless otherwise mentioned, the terms “Gd”,“gadolinium” and “Gd³+” are used without distinction to denote the Gd³⁺ion. By extension, it may also be a source of free gadolinium, such asgadolinium chloride (GdCl₃) or gadolinium oxide (Gd₂O₃).

In the present invention, the term “free Gd” denotes the non-complexedforms of gadolinium, which are preferably available for complexation. Itis typically the Gd³⁺ ion dissolved in water. By extension, it may alsobe a source of free gadolinium, such as gadolinium chloride (GdCl₃) orgadolinium oxide.

-   -   Step a)

In this step, a complexation reaction takes place between the hexaacidof formula (III) and gadolinium, which makes it possible to obtain thehexaacid gadolinium complex of formula (I) as defined previously.

According to a particular embodiment, step a) comprises the reactionbetween the hexaacid of formula (III) and a source of free Gd in water.

In a preferred embodiment, the source of free Gd is GdCl₃ or Gd₂O₃,preferably Gd₂O₃.

Preferably, the reagents used in step a), i.e. the source of gadolinium(typically gadolinium oxide), the hexaacid of formula (III) and water,are as pure as possible, notably as regards the metal impurities.

Thus, the source of gadolinium will advantageously be gadolinium oxide,preferably with a purity of greater than 99.99% and even more preferablygreater than 99.999%.

The water used in the process preferably comprises less than 50 ppm ofcalcium, more preferably less than 20 ppm and most preferably less than15 ppm of calcium. Generally, the water used in the process is deionizedwater, water for injection (injection-grade water) or purified water.

Advantageously, the amounts of the reagents (the hexaacid of formula(III) and gadolinium) used in this step a) correspond to, or are closeto, stoichiometric proportions, as dictated by the balance equation ofthe complexation reaction which takes place during this step.

The term “close to stoichiometric proportions” means that the differencebetween the molar proportions in which the reagents are introduced andthe stoichiometric proportions is less than 15%, notably less than 10%,preferably less than 8%.

Gadolinium may notably be introduced in slight excess relative to thestoichiometric proportions. The ratio of the amount of materialintroduced as gadolinium to the amount of material introduced ashexaacid of formula (III) is then greater than 1, but typically lessthan 1.15, notably less than 1.10, advantageously less than 1.08. Inother words, the amount of gadolinium introduced is greater than 1equivalent (eq.), but typically less than 1.15 eq., notably less than1.10 eq., advantageously less than 1.08 eq., relative to the amount ofhexaacid of formula (III) introduced, which itself corresponds to 1equivalent. In the preferred embodiment in which the source of freegadolinium is Gd₂O₃, the amount of Gd₂O₃ introduced is then typicallygreater than 0.5 eq., but less than 0.575 eq., notably less than 0.55eq., advantageously less than 0.54 eq., relative to the amount ofhexaacid of formula (III) introduced (1 eq.).

According to a particular embodiment, step a) comprises the followingsuccessive steps:

-   -   a1) Preparation of an aqueous solution of hexaacid of formula        (III), and    -   a2) Addition, to the aqueous solution obtained in step al), of a        source of free gadolinium.

In this embodiment, the content of hexaacid of formula (III) in theaqueous solution prepared in step al) is typically between 10% and 60%,notably between 15% and 45%, preferably between 20% and 35%,advantageously between 25% and 35% and even more advantageously between25% and 30% by weight relative to the total weight of the aqueoussolution.

Preferentially, steps a) and b) are performed according to a one-potembodiment, i.e. in the same reactor and without an intermediate step ofisolation or purification.

Thus, in this preferred embodiment, the hexaacid gadolinium complex offormula (I) formed in step a) is directly subjected to the isomerizationstep b) without being isolated or purified, and in the same reactor asthat used for step a).

-   -   Step b)

The hexaacid gadolinium complex of formula (I) formed by thecomplexation reaction between the hexaacid of formula (III) andgadolinium in step a) is initially obtained in the form of a mixture ofdiastereoisomers.

Step b) aims at enriching the mixture of diastereoisomers in the isomersI-RRR and I-SSS, to obtain the diastereoisomerically enriched hexaacidgadolinium complex of formula (I) constituted of at least 85%, notablyof at least 90%, in particular of at least 95%, preferably of at least97%, advantageously of at least 98%, more advantageously of at least 99%of a diastereoisomeric excess comprising the mixture of the isomersI-RRR and I-SSS.

In the context of the present invention, the term “diastereoisomericexcess” is intended to denote, as regards the hexaacid gadoliniumcomplex of formula (I), the fact that said complex is predominantlypresent in the form of an isomer or group of isomers chosen from thediastereoisomers I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR andI-SRS. Said diastereoisomeric excess is expressed as a percentage andcorresponds to the amount represented by the predominant isomer or groupof isomers relative to the total amount of the hexaacid gadoliniumcomplex of formula (I). It is understood that this percentage may be oneither a molar or mass basis, since isomers have, by definition, thesame molar mass.

Preferably, said diastereoisomeric excess is constituted of at least70%, notably of at least 80%, advantageously of at least 90%, preferablyof at least 95% of the mixture of isomers I-RRR and I-SSS.

Advantageously, said diastereoisomeric excess consists of the mixture ofisomers I-RRR and I-SSS.

The inventors have in fact discovered that factors such as the pH andthe temperature of the solution of hexaacid gadolinium complex offormula (I) obtained on conclusion of step a) have an influence on theratio in which the various isomers of the complex of formula (I) arepresent in the mixture of diastereoisomers. Over time, the mixture tendsto become enriched in a group of isomers comprising the isomers whichare, surprisingly, the most thermodynamically stable but also the mostchemically stable, in this instance the isomers I-RRR and I-SSS.

The term “mixture of isomers I-RRR and I-SSS” also covers, by extension,the case where only one of the isomers, whether it be I-RRR or I-SSS, ispresent.

However, in a preferred embodiment, the isomers I-RRR and I-SSS arepresent in said mixture in a ratio of between 65/35 and 35/65, notablybetween 60/40 and 40/60, in particular between 55/45 and 45/55.Advantageously, the mixture of isomers I-RRR/I-SSS is a racemic (50/50)mixture.

Step b) of isomerization of the hexaacid gadolinium complex of formula(I) in an aqueous solution is typically performed at a pH of between 2and 4, notably between 2 and 3, advantageously between 2.2 and 2.8.

The pH is preferentially adjusted with an acid, preferably an inorganicacid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid or phosphoric acid, for example with hydrochloric acid.

It is entirely surprising that, under such pH conditions, enrichment ofthe mixture in particular isomers, in this instance the isomers I-RRRand I-SSS, takes place, since it is known in the art that gadoliniumchelates are characterized by low kinetic inertia in acidic medium.Indeed, the higher the concentration of H⁺ ions in the medium, thegreater the probability that a proton is transferred onto one of thedonor atoms of the ligand, thus bringing about dissociation of thecomplex. Consequently, a person skilled in the art would have expectedthat placing the hexaacid gadolinium complex of formula (I) in anaqueous solution at a pH of between 2 and 4 would bring aboutdissociation of said complex, rather than its isomerization into I-RRRand I-SSS.

It should be noted that the pH range recommended by EP 1 931 673 for thecomplexation of the hexaacid of formula (III), namely 5.0-6.5, does notmake it possible to obtain the complex of formula (I) enriched in itsisomers I-RRR and I-SSS.

Step b) is typically performed at a temperature of between 80° C. and130° C., notably between 90° C. and 125° C., preferably between 98° C.and 122° C., advantageously between 100° C. and 120° C., typically for atime of between 10 hours and 72 hours, notably between 10 hours and 60hours, advantageously between 12 hours and 48 hours.

Contrary to all expectations, such temperature conditions, which,combined with the abovementioned pH conditions, should favour theinstability of the gadolinium chelate, do not result in itsdecomplexation or in the formation of any other impurity, but in itsisomerization into I-RRR and I-SSS.

In one particular embodiment, the aqueous solution of step b) comprisesacetic acid. Step b) is then advantageously performed at a temperatureof between 100° C. and 120° C., notably between 110° C. and 118° C.,typically for a time of between 12 hours and 48 hours, notably between20 hours and 30 hours, in particular between 24 hours and 26 hours.

The acetic acid is preferably added before the heating of the solutionof hexaacid gadolinium complex of formula (I) obtained in step a) in anamount such that the acetic acid content is between 25% and 75%, notablybetween 40% and 50% by mass relative to the mass of hexaacid of formula(III) used in step a).

When the aqueous solution is heated to a temperature advantageouslybetween 100° C. and 120° C., typically between 110° C. and 118° C.,acetic acid is added gradually as the water evaporates, so as tomaintain a constant volume of solution.

According to a preferred embodiment, on conclusion of step b), thediastereoisomerically enriched complex is isolated by crystallization,preferably by crystallization by seeding.

In this embodiment, step b) comprises the following successive steps:

-   -   b1) Isomerization by heating the hexaacid gadolinium complex of        formula (I) in an aqueous solution at a pH of between 2 and 4 to        obtain a diastereoisomerically enriched complex constituted of        at least 80% of the diastereoisomeric excess comprising the        mixture of the isomers I-RRR and I-SSS of said hexaacid        gadolinium complex of formula (I), and    -   b2) Isolation by crystallization of said diastereoisomerically        enriched complex, preferably by crystallization by seeding.

The crystallization step b2) aims firstly at removing any impuritiespresent in the aqueous solution, which may result from previous steps,so as to obtain a decolourized product of higher purity, in the form ofcrystals, and secondly at continuing the diastereoisomeric enrichment ofthe hexaacid gadolinium complex of formula (I), so as to obtain adiastereoisomeric excess comprising the mixture of the isomers I-RRR andI-SSS of said complex which is higher than that obtained on conclusionof step b1). Indeed, the isomers I-RRR and I-SSS of the hexaacid complexof formula (I) crystallize from water. On the other hand, the hexaacidgadolinium complex of formula (I) not enriched in said isomers does notcrystallize.

The fact that the isomers I-RRR and I-SSS, in which the complex tends tobecome enriched in the course of step b) (and, contrary to allexpectations, in the light of the conditions under which it isperformed), are the only isomers of the complex to crystallize fromwater is an entirely unexpected result. The isomerization andcrystallization thus contribute synergistically towards the enrichmentin isomers I-RRR and I-SSS and consequently towards the overallefficiency of the process according to the invention.

Moreover, it should be noted that crystallization from water of theisomers of interest of the hexaacid gadolinium complex of formula (I)makes it possible to avoid an addition of solvent as described inExample 7 of EP 1 931 673, which involves a step of precipitation fromethanol of the trisodium salt of said complex.

Step b2) is advantageously performed at a temperature of between 10° C.and 70° C., notably between 30° C. and 65° C., in particular between 35°C. and 60° C.

According to one variant, after lowering the temperature of the aqueoussolution, so that it is within the ranges indicated above, thecrystallization process is induced by seeding. “Crystallization byseeding”, also known as “crystallization by priming”, comprises theintroduction into the reactor in which the crystallization is performed(also known as the crystallization vessel) of a known amount ofcrystals, known as “seed” or “primer”. This makes it possible to reducethe crystallization time. Crystallization by seeding is well known tothose skilled in the art. In the process according to the invention,seeding using a primer, in the present instance crystals ofdiastereoisomerically enriched hexaacid gadolinium complex of formula(I) added to the aqueous solution of the diastereoisomerically enrichedcomplex whose temperature has been lowered beforehand, makes it possibleto obtain nucleation, and thus to initiate the crystallization. Theduration of the crystallization by seeding is advantageously between 2hours and 20 hours and preferably between 6 hours and 18 hours;typically, it is 16 hours.

The crystals of diastereoisomerically enriched hexaacid gadoliniumcomplex of formula (I) are then typically isolated by filtration anddrying, by means of any technique well known to those skilled in theart.

Advantageously, the degree of purity of the diastereoisomericallyenriched hexaacid gadolinium complex of formula (I) isolated onconclusion of step b2) is greater than 95%, notably greater than 98%,advantageously greater than 99%, said degree of purity being expressedas a mass percentage of the complex of formula (I) relative to the totalmass obtained on conclusion of step b2).

In a particular embodiment, the diastereoisomerically enriched complexfrom step b) isolated by crystallization is again purified byrecrystallization, to obtain a diastereoisomerically enriched andpurified complex.

In this embodiment, step b) comprises, besides the successive steps b1)and b2) described previously, a step b3) of purification byrecrystallization of the isolated diastereoisomerically enrichedhexaacid gadolinium complex of formula (I).

The recrystallization step b3) aims, like the crystallization step b2),firstly at obtaining a product of higher purity, and secondly atcontinuing the diastereoisomeric enrichment of the hexaacid gadoliniumcomplex of formula (I), so as to obtain a diastereoisomeric excesscomprising the mixture of the isomers I-RRR and I-SSS of said complexwhich is higher than that obtained on conclusion of step b2).

Step b3) typically comprises the following successive substeps:

-   -   suspension of the diastereoisomerically enriched hexaacid        gadolinium complex of formula (I) isolated in step b2) in        aqueous solution, preferably in water,    -   dissolution of said complex by heating to a temperature        advantageously between 80° C. and 120° C., for example to 100°        C.,    -   recrystallization, preferably by seeding, at a temperature        advantageously between 10° C. and 90° C., notably between 20° C.        and 87° C., in particular between 55° C. and 85° C., typically        for a time of between 2 hours and 20 hours, notably between 6        hours and 18 hours, and    -   isolation of the crystals of diastereoisomerically enriched and        purified hexaacid gadolinium complex of formula (I), for example        by filtration and drying.

The degree of purity of the purified diastereoisomerically enrichedhexaacid gadolinium complex of formula (I) isolated on conclusion ofstep b3) is typically greater than 98%, notably greater than 99%,advantageously greater than 99.5%, said degree of purity being expressedas a mass percentage of the complex of formula (I) relative to the totalmass obtained on conclusion of step b2).

In another embodiment, the diastereoisomerically enriched complex fromstep b) is further enriched by selective decomplexation of thediastereoisomers of the complex of formula (I) other than thediastereoisomers I-RRR and I-SSS, i.e. by selective decomplexation ofthe diastereoisomers I-RSS, I-SRR, I-RSR, I-SRS, I-RRS and I-SSR.

In this embodiment, step b) comprises, besides the successive steps b1)and b2) described previously, a step b4) of selective decomplexation ofthe diastereoisomers of the complex of formula (I) other than thediastereoisomers I-RRR and I-SSS. In this variant, step b) may alsocomprise step b3) described previously, said step b3) being performedbetween steps b2) and b4), or after b4).

The selective decomplexation step b4) is directed towards continuing thediastereoisomeric enrichment of the hexaacid gadolinium complex offormula (I), so as to obtain a diastereoisomeric excess comprising themixture of the isomers I-RRR and I-SSS of said complex which is higherthan that obtained on conclusion of step b2) or on conclusion of stepb3), when said step is performed prior to step b4).

Step b4) typically comprises the following successive substeps:

-   -   suspension of the diastereoisomerically enriched hexaacid        gadolinium complex of formula (I) isolated in step b2) or in        step b3) in water,    -   addition of a base, for example sodium hydroxide,    -   heating to a temperature advantageously between 30° C. and 60°        C., notably between 35° C. and 55° C., for example at 40° C.,        typically for a time of between 2 hours and 20 hours, notably        between 10 hours and 18 hours,    -   cooling to a temperature advantageously between 10° C. and 30°        C., for example to 30° C., and    -   isolation of the diastereoisomerically enriched and purified        hexaacid gadolinium complex of formula (I), for example by        filtration and drying.

Step b4) is made possible by the fact that the isomers I-RRR and I-SSSare the most stable in basic medium. Such basic conditions promote theformation of gadolinium hydroxide, and consequently the decomplexationof the least stable isomers. Thus, it should be noted that,surprisingly, the isomers I-RRR and I-SSS are more stable both in acidicmedium, which allows the isomerization step b1), and in basic medium,which allows the selective decomplexation step b4).

In a preferred embodiment, the diastereoisomerically enriched complexobtained on conclusion of step b) according to any one of the variantsdescribed above has at least 85%, notably at least 90%, in particular atleast 95%, preferably at least 97%, advantageously at least 98%, moreadvantageously at least 99% of the diastereoisomeric excess comprisingthe mixture of isomers I-RRR and I-SSS.

Preferably, said diastereoisomeric excess is constituted of at least70%, notably of at least 80%, advantageously of at least 90%, preferablyof at least 95% of the mixture of isomers I-RRR and I-SSS.

Advantageously, said diastereoisomeric excess consists of the mixture ofisomers I-RRR and I-SSS.

The term “mixture of isomers I-RRR and I-SSS” also covers, by extension,the case where only one of the isomers, whether it be I-RRR or I-SSS, ispresent. However, the term “mixture of isomers I-RRR and I-SSS”preferentially denotes all the cases in which each of the isomers I-RRRand I-SSS is present in a variable but non-zero amount.

In a preferred embodiment, the isomers I-RRR and I-SSS are present insaid mixture in a ratio of between 65/35 and 35/65, notably between60/40 and 40/60, in particular between 55/45 and 45/55. Advantageously,the mixture of isomers I-RRR/I-SSS is a racemic (50/50) mixture.

-   -   Step c)

Step c) aims at forming the complex of formula (II) from its precursor,the diastereoisomerically enriched hexaacid gadolinium complex offormula (I) obtained in step b).

During this step, the three carboxylic acid functions of the hexaacidcomplex of formula (I) borne by the carbon atoms located in the γposition on the side chains of the complex, relative to the nitrogenatoms of the macrocycle on which said side chains are grafted, areconverted into amide functions, via an amidation reaction with3-amino-1,2-propanediol, in racemic or enantiomerically pure form,preferably in racemic form.

This amidation reaction does not modify the absolute configuration ofthe three asymmetric carbon atoms located in the α position on the sidechains, relative to the nitrogen atoms of the macrocycle onto which saidside chains are grafted. Consequently, step c) makes it possible toobtain the complex of formula (II) with a diastereoisomeric excesscomprising a mixture of the isomers II-RRR and II-SSS that is identicalto the diastereoisomeric excess comprising a mixture of the isomersI-RRR and I-SSS with which is obtained the diastereoisomericallyenriched hexaacid gadolinium complex of formula (I) obtained onconclusion of step b), which is at least 80%.

In a preferred embodiment, the complex of formula (II) obtained onconclusion of step c) has at least 85%, notably at least 90%, inparticular at least 92%, preferably at least 94%, advantageously atleast 97%, more advantageously at least 99% of the diastereoisomericexcess comprising the mixture of isomers II-RRR and II-SSS.

Preferably, said diastereoisomeric excess is constituted of at least70%, notably of at least 80%, advantageously of at least 90%, preferablyof at least 95% of the mixture of isomers II-RRR and II-SSS.

Advantageously, said diastereoisomeric excess consists of the mixture ofisomers II-RRR and II-SSS.

The term “mixture of isomers II-RRR and II-SSS” also covers, byextension, the case where only one of the isomers, whether it be II-RRRor II-SSS, is present. However, the term “mixture of isomers II-RRR andII-SSS” preferentially denotes all the cases in which each of theisomers II-RRR and II-SSS is present in a variable but non-zero amount.

In a preferred embodiment, the isomers II-RRR and II-SSS are present insaid mixture in a ratio of between 65/35 and 35/65, notably between60/40 and 40/60, in particular between 55/45 and 45/55. Advantageously,the isomers II-RRR and II-SSS are present in the mixture in a 50/50ratio.

The amidation reaction may be performed according to any method that iswell known to those skilled in the art, notably in the presence of anagent for activating carboxylic acid functions and/or by acid catalysis.

It may notably be performed according to the methods described in EP 1931 673, notably in paragraph [0027] of said patent.

In one particular embodiment, step c) comprises the activation of thecarboxylic acid (—COOH) functions of the hexaacid complex of formula (I)borne by the carbon atoms located in the γ position on the side chainsof the complex, relative to the nitrogen atoms of the macrocycle onwhich said side chains are grafted, in the form of functionalderivatives including a carbonyl (C═O) group, which are such that thecarbon atom of the carbonyl group is more electrophilic than the carbonatom of the carbonyl group of the carboxylic acid functions. Thus,according to this particular embodiment, said carboxylic acid functionsmay notably be activated in the form of ester, acyl chloride or acidanhydride functions, or in any activated form that can lead to an amidebond. The activated forms that can lead to an amide bond are well knownto those skilled in the art and may be obtained, for example, by the setof methods known in peptide chemistry for creating a peptide bond.Examples of such methods are given in the publication Synthesis ofpeptides and peptidomimetics volume E22a, pages 425-588, Houben-Weyl etal., Goodman Editor, Thieme-Stuttgart-New York (2004), and, among theseexamples, mention may be made notably of the methods of activation ofcarboxylic acids via an azide (acyl azide), for example via the actionof a reagent such as diphenylphosphoryl azide (commonly referred to bythe abbreviation DPPA), the use of carbodiimides alone or in thepresence of catalysts (for example N-hydroxysuccinimide and derivativesthereof), the use of a carbonyldiimidazole (1,1′-carbonyldiimidazole,CDI), the use of phosphonium salts such asbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(commonly referred to by the abbreviation BOP), or else uroniums such as2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(commonly referred to by the abbreviation HBTU).

Preferably, step c) comprises the activation of the abovementionedcarboxylic acid (—COOH) functions in the form of ester, acyl chloride oracid anhydride functions.

This embodiment is preferred to peptide coupling by activation of thecarboxylic acid function using a coupling agent such as EDCl/HOBT asdescribed in EP 1 931 673. Indeed, such coupling leads to the formationof one equivalent of 1-ethyl-3-[3-(dimethylamino)propyl]urea, which mustbe removed, notably by chromatography on silica or by liquid/liquidextraction by adding a solvent. Independently of the increasedcomplexity of the process caused by such an additional step, the use ofsuch purification methods is not desirable, as discussed previously.Furthermore, the use of HOBT is in itself problematic, since it is anexplosive product.

For the purposes of the present invention, the term “ester function” isintended to denote a —C(O)O— group. It may in particular be a group—C(O)O-R₁, in which R₁ corresponds to a (C₁-C₆)alkyl group.

For the purposes of the present invention, the term “(C₁-C₆)alkyl group”means a linear or branched, saturated hydrocarbon-based chain containing1 to 6 and preferably 1 to 4 carbon atoms. Examples that may bementioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl and hexyl groups.

For the purposes of the present invention, the term “acyl chloridefunction”, also known as “acid chloride function” is intended to denotea —CO—Cl group.

For the purposes of the present invention, the term “acid anhydridefunction” is intended to denote a —CO—O—CO— group. It may in particularbe a group —CO—O—CO—R₂, in which R₂ corresponds to a (C₁-C₆)alkyl group.

The reactions for converting a carboxylic acid function into an ester,acyl chloride or acid anhydride function are well known to a personskilled in the art, who will be able to perform them according to anyusual method with which he is familiar.

The complex of formula (II) is then obtained by aminolysis of thecarboxylic acid functions activated in the form of ester, acyl chlorideor acid anhydride functions, notably esters or acid anhydrides,preferably esters, by reaction with 3-amino-1,2-propanediol, in racemicor enantiomerically pure form, preferably in racemic form.

Preferentially, the steps of activating the carboxylic acid functionsand of aminolysis are performed according to a one-pot embodiment, i.e.in the same reactor and without an intermediate step of isolation orpurification of the intermediate including the carboxylic acid functionsactivated in the form of ester, acyl chloride or acid anhydridefunctions, notably esters or acid anhydrides, preferably esters.

According to a particular embodiment, step c) comprises the followingsuccessive steps:

-   -   c1) formation of an activated complex of formula (VII),

-   -   in which Y represents a chlorine atom, a group —OR₁ or        —O—C(O)—R₂; preferably, Y represents a group —OR₁ or —O—C(O)—R₂,        with R₁ and R₂ corresponding, independently of each other, to a        (C₁-C₆)alkyl group, and    -   c2) aminolysis of the activated complex of formula (VII) with        3-amino-1,2-propanediol.

As will be clearly apparent to a person skilled in the art, the reactionfor formation of the activated complex of formula (VII) does not modifythe absolute configuration of the three asymmetric carbon atoms locatedin the α position on the side chains, relative to the nitrogen atoms ofthe macrocycle onto which said side chains are grafted. Consequently,step c1) makes it possible to obtain the activated complex of formula(VII) with a diastereoisomeric excess comprising a mixture of theisomers VII-RRR and VII-SSS, of formulae (VII-RRR) and (VII-SSS)represented below, that is identical to the diastereoisomeric excesscomprising a mixture of the isomers I-RRR and I-SSS with which isobtained the diastereoisomerically enriched hexaacid gadolinium complexof formula (I) obtained on conclusion of step b), which is at least 80%.

In the case where Y represents a chlorine atom, step c1) is typicallyperformed by reaction between the diastereoisomerically enrichedhexaacid gadolinium complex of formula (I) obtained in step b) andthionyl chloride (SOCl₂).

In the case where Y represents an —O—C(O)—CH₃ group, step c1) istypically performed by reaction between the diastereoisomericallyenriched hexaacid gadolinium complex of formula (I) obtained in step b)and acetyl chloride.

In an advantageous embodiment, step c) comprises the activation of theabovementioned carboxylic acid (—COOH) functions in the form of esterfunctions.

According to this embodiment, step c) may more particularly comprise thefollowing successive steps:

-   -   c1) formation of a triester of formula (VIII),

-   -   in which R₁ represents a (C₁-C₆)alkyl group, and    -   c2) aminolysis of the triester of formula (VIII) with        3-amino-1,2-propanediol.

Step c1) is typically performed in the alcohol of formula R₁OH, whichacts both as solvent and as reagent, in the presence of an acid such ashydrochloric acid.

Step c2) is also typically performed in the alcohol of formula R₁OH, inthe presence of an acid such as hydrochloric acid.

In a first stage, the hexaacid gadolinium complex of formula (I) and thealcohol R₁OH are placed in the reactor. The reaction medium is thencooled to a temperature below 10° C., notably below 5° C., typically to0° C., and an acidic solution of the alcohol R₁OH, typically ofhydrochloric acid in R₁OH, is then gradually added. The reaction mediumis kept stirring at room temperature (i.e. at a temperature between 20and 25° C.) for a time typically greater than 5 hours, preferablybetween 10 hours and 20 hours. The reaction medium is cooled to atemperature below 10° C., notably between 0° C. and 5° C., prior to stepc2).

Thus, steps c1) and c2) may be readily performed according to a one-potembodiment. Advantageously, the triester of formula (VII) is notisolated between steps c1) and c2).

However, in order to promote the aminolysis reaction, in step c2), thealcohol of formula R₁OH is preferably removed by vacuum distillation.

For the purposes of the present invention, the term “vacuumdistillation” means the distillation of a mixture performed at apressure of between 10 and 500 mbar, notably between 10 and 350 mbar,preferably between 10 and 150 mbar, in particular between 50 and 100mbar.

Similarly, in order to promote the aminolysis reaction, in step c2),3-amino-1,2-propanediol is introduced in large excess. Typically, thematerial amount of 3-amino-1,2-propanediol introduced is greater than 4eq., notably greater than 7 eq., advantageously greater than 10 eq.,relative to the material amount of diastereoisomerically enrichedhexaacid gadolinium complex of formula (I) initially introduced in stepc), which itself corresponds to 1 equivalent.

Surprisingly, despite the acidic conditions typically employed in stepsc1) and c2), which should increase the kinetic instability of thegadolinium complexes, no decomplexation or isomerization of the triesterof formula (VIII) is observed. The desired triamide is obtained with avery good degree of conversion and the absolute configuration of thethree asymmetric carbon atoms located in the α position on the sidechains, relative to the nitrogen atoms of the macrocycle, is conserved.

Moreover, it should be noted that, in general, amidation reactions bydirect reaction between an ester and an amine are very sparinglydescribed in the literature (see on this subject K. C. Nadimpally etal., Tetrahedron Letters, 2011, 52, 2579-2582).

In a preferred embodiment, step c) comprises the following successivesteps:

-   -   c1) formation of a methyl triester of formula (IV),

-   -   notably by reaction in methanol in the presence of an acid such        as hydrochloric acid, and    -   c2) aminolysis of the methyl triester of formula (IV) with        3-amino-1,2-propanediol, notably in methanol in the presence of        an acid such as hydrochloric acid.

Advantageously, the methyl triester of formula (IV) is not isolatedbetween steps c1) and c2).

In a preferred embodiment, in step c2), the methanol is removed byvacuum distillation, until a temperature typically greater than 55° C.,notably between 60° C. and 65° C. is reached, and the reaction medium ismaintained at this temperature under vacuum for a time typically greaterthan 5 hours, notably between 10 hours and 20 hours, before being cooledto room temperature and diluted with water.

The present invention encompasses all the combinations of theparticular, advantageous or preferred embodiments described above inconnection with each step of the process.

-   -   Preparation of the hexaacid of formula (III)

The hexaacid of formula (III), which participates in step a) of theprocess for preparing the complex of formula (II) according to theinvention, may be prepared according to any method already known andnotably according to the methods described in EP 1 931 673. However,according to a preferred embodiment, the hexaacid of formula (III) isobtained by alkylation of the pyclene of formula (V):

with a compound of formula R₃OOC—CHG_(p)-(CH₂)₂—COOR₄ (IX),

in which:

-   -   R₃ and R₄ represent, independently of each other, a (C₃-C₆)alkyl        group, notably a (C₄-C₆)alkyl group such as a butyl, isobutyl,        sec-butyl, tert-butyl, pentyl or hexyl group, and    -   G_(p) represents a leaving group such as a tosylate or triflate        group, or a halogen atom, preferably a bromine atom,

to obtain the hexaester of formula (X)

followed by a hydrolysis step, leading to said hexaacid of formula(III).

In a preferred embodiment, R₃ and R₄ are identical.

According to an advantageous embodiment, the hexaacid of formula (III)is obtained by alkylation of the pyclene of formula (V):

with dibutyl 2-bromoglutarate, to obtain the butyl hexaester of formula(VI):

followed by a hydrolysis step, leading to said hexaacid of formula(III).

The dibutyl 2-bromoglutarate used is in racemic or enantiomerically pureform, preferably in racemic form.

The use of dibutyl 2-bromoglutarate is particularly advantageous, incomparison with the use of ethyl 2-bromoglutarate described in EP 1 931673. Indeed, commercial diethyl 2-bromoglutarate is a relativelyunstable compound, which degrades over time and under the effect of thetemperature. More precisely, this ester has a tendency to becomehydrolysed or to cyclize and thus to lose its bromine atom. Attempts topurify commercial diethyl 2-bromoglutarate, or to develop new syntheticroutes for obtaining it with improved purity, and thus to prevent itsdegradation, were unsuccessful.

The alkylation reaction is typically performed in a polar solvent,preferably in water, in particular in deionized water, advantageously inthe presence of a base such as potassium or sodium carbonate.

The use of water is preferred notably to that of acetonitrile, describedin EP 1 931 673, for obvious reasons.

The reaction is advantageously performed at a temperature of between 40°C. and 80° C., typically between 50° C. and 70° C. and notably between55° C. and 60° C., for a time of between 5 hours and 20 hours, inparticular between 8 hours and 15 hours.

The hydrolysis step is advantageously performed in the presence of anacid or a base, advantageously a base such as sodium hydroxide. Thehydrolysis solvent may be water, an alcohol such as ethanol, or awater/alcohol mixture. This step is advantageously performed at atemperature of between 40° C. and 80° C., typically between 40° C. and70° C. and notably between 50° C. and 60° C., typically for a time ofbetween 10 hours and 30 hours, in particular between 15 hours and 25hours.

Process for Purifying the Complex of Formula (II)

The present invention furthermore relates to a process for purifying thecomplex of formula (II) below:

with at least 80% of a diastereoisomeric excess comprising a mixture ofisomers II-RRR and II-SSS of formulae:

comprising:

-   -   1) the combination of the following two steps:        -   1b) passage through ion-exchange resin(s), and        -   1c) ultrafiltration of said complex, and    -   2) isolation of the purified complex thus obtained in solid        form.

Advantageously, said complex of formula (II) having at least 80%,preferentially at least 85%, notably at least 90%, in particular atleast 95%, more particularly at least 97%, preferably at least 98% andadvantageously at least 99% of a diastereoisomeric excess comprising amixture of isomers II-RRR and II-SSS was obtained previously accordingto the preparation process described previously.

In a preferred embodiment, the diastereoisomerically enriched complex onwhich the purification process is performed has at least 85%, notably atleast 90%, in particular at least 92%, preferably at least 94%,advantageously at least 97%, more advantageously at least 99% of thediastereoisomeric excess comprising the mixture of isomers II-RRR andII-SSS.

Preferably, said diastereoisomeric excess is constituted of at least70%, notably of at least 80%, advantageously of at least 90%, preferablyof at least 95% of the mixture of isomers II-RRR and II-SSS.

Advantageously, said diastereoisomeric excess consists of the mixture ofisomers II-RRR and II-SSS.

The term “mixture of isomers II-RRR and II-SSS” also covers, byextension, the case where only one of the isomers, whether it be II-RRRor II-SSS, is present. However, the term “mixture of isomers II-RRR andII-SSS” preferentially denotes all the cases in which each of theisomers II-RRR and II-SSS is present in a variable but non-zero amount.

In a preferred embodiment, the isomers II-RRR and II-SSS are present insaid mixture in a ratio of between 65/35 and 35/65, notably between60/40 and 40/60, in particular between 55/45 and 45/55. Advantageously,the isomers II-RRR and II-SSS are present in the mixture in a 50/50ratio.

-   -   Combination of steps 1b) and 1c)

Steps 1b) and 1c) are directed towards purifying the complex of formula(II) by removing the impurities that may be present due to itsproduction process.

Said impurities may notably comprise 3-amino-1,2-propanediol and/or adicoupled impurity.

Indeed, 3-amino-1,2-propanediol may be present in the final productobtained during the implementation of a process for preparing thecomplex of formula (II), typically when the complex of formula (II) isobtained by amidation starting with the complex of formula (I) and3-amino-1,2-propanediol. This is notably the case for the process forpreparing the complex of formula (II) according to the invention. Asdetailed previously, the amidation reaction may comprise the activationof the three carboxylic acid functions borne by the carbon atoms locatedin the γ position on the side chains of the complex of formula (I),relative to the nitrogen atoms of the macrocycle on which said sidechains are grafted, followed by aminolysis of the activated carboxylicacid functions by reaction with 3-amino-1,2-propanediol. The3-amino-1,2-propanediol is then advantageously used in excess, so as toensure good conversion into amide functions of the three activatedcarboxylic acid functions.

The term “dicoupled impurity” is intended to denote a complex of formula(II-dc-a), (II-dc-b), (II-dc-c) represented below, or a mixture thereof:

The dicoupled impurity may notably result from the hydrolysis reactionof an amide function of the complex of formula (II). It may also resultfrom incomplete activation of the carboxylic acid functions of thecomplex of formula (I) (activation of two out of the three functions) orfrom incomplete aminolysis of the activated carboxylic acid functions(aminolysis of two out of the three functions), when the process forpreparing the complex of formula (II) involves such steps. This isnotably the case for the process for preparing the complex of formula(II) according to the invention.

-   -   Step 1 b) corresponds to the passage of the        diastereoisomerically enriched complex of formula (II) as        described previously through ion-exchange resin(s).

For the purposes of the present invention, the term “ion-exchange resin”means a solid material which is generally in the form of beads composedof a polymer matrix onto which are grafted positively charged functionalgroups (anionic resin) or negatively charged functional groups (cationicresin), which will make it possible, respectively, to trap anions orcations by adsorption. The adsorption of the anions or cations onto theresin proceeds via ion exchange between the counterions of thefunctional groups initially present so as to ensure the electricalneutrality of the resin, and the anions or cations intended to betrapped.

Step 1 b) involves placing an aqueous solution of thediastereoisomerically enriched complex of formula (II) in contact with astrong anionic resin. The water used is preferably a purified water.

Said strong anionic resin typically includes, as exchanging functionalgroups, ammonium groups (N(RR′R″)⁺, in which R, R′ and R″ are identicalor different (C₁-C₆)alkyl groups). Mention may notably be made of theresin Amberlite® FPA900 sold by Dow Chemical, advantageously in HO⁻form.

Passage through the strong anionic resin makes it possible to at leastpartly remove the dicoupled impurities.

Step 1 b) may also involve placing an aqueous solution of thediastereoisomerically enriched complex of formula (II) in contact with aweak cationic resin. The water used is preferably a purified water.

Said weak cationic resin typically includes, as exchanging functionalgroups, carboxylate groups (CO₂ ⁻). Mention may notably be made of theresin IMAC® HP336 sold by Dow Chemical, advantageously in H⁺ form.

Passage through the weak cationic resin makes it possible to at leastpartly remove the 3-amino-1,2-propanediol, and the possible Gd³⁺residues.

It should be noted that step 1b) of passage through ion-exchangeresin(s) is made possible by the improved stability of thediastereoisomerically enriched complex of formula (II) according to theinvention, the integrity of which is consequently preserved during thisstep.

-   -   Step 1c) corresponds to ultrafiltration of the        diastereoisomerically enriched complex of formula (II) as        described previously.

In the present invention, the term “ultrafiltration” means a method offiltration through a mesoporous semi-permeable membrane, the pores ofwhich generally have a diameter of between 1 and 100 nm, in particularbetween 2 and 50 nm, notably between 10 and 50 nm (mesopores), under theeffect of forces such as pressure gradients, typically between 1 and 10bar, and optionally concentration gradients. It is thus a process ofmembrane separation via which particles in solution or in suspensionwhose size is greater than that of the pores are retained by themembrane and separated from the liquid mixture which contained them.

In the context of the purification process according to the invention,ultrafiltration is particularly advantageous for removing endotoxins.

Advantageously, the ultrafiltration membrane used in step 1c) has acut-off threshold of less than 100 kD, notably less than 50 kD, inparticular less than 25 kD, typically a cut-off threshold of 10 kD.

Preferably, in step 1c), the transmembrane pressure is between one and 5bar, in particular between 2.25 and 3.25 bar.

-   -   In one particular embodiment, steps 1b) and 1c) are also        combined with a nanofiltration step 1a).

In the present invention, the term “nanofiltration” means a method offiltration through a porous semi-permeable membrane, the pores of whichgenerally have a diameter of between 0.1 and 100 nm, in particularbetween 0.1 and 20 nm, notably between 1 and 10 nm, under the effect offorces such as pressure gradients, typically between 1 and 50 bar, andoptionally concentration gradients. It is thus a process of membraneseparation via which particles in solution or in suspension whose sizeis greater than that of the pores are retained by the membrane andseparated from the liquid mixture which contained them.

The nanofiltration step 1a) makes it possible to remove the greater partof the excess 3-amino-1,2-propanediol (optionally in salt form, inparticular hydrochloride, or in the form of derivatives, notably theacetamide derivative) and the mineral salts.

In this particular embodiment, the nanofiltration step may be performeddirectly on the crude diastereoisomerically enriched complex of formula(II) as obtained according to the preparation process describedpreviously. It is notably not necessary to precipitate thediastereoisomerically enriched complex of formula (II) preparedpreviously by adding solvent.

Advantageously, the nanofiltration membrane used in step 1a) has acut-off threshold of less than 1 kD, notably less than 500 daltons, inparticular less than 300 daltons, typically a cut-off threshold of 200daltons.

Preferably, in step 1a), the transmembrane pressure is between 10 and 40bar, in particular between 2 and 30 bar.

In particular, the temperature of the solution of the complex of formula(II) subjected to ultrafiltration in step 1a) is between 20 and 40° C.,notably between 25 and 35° C.

In one alternative of this particular embodiment, step 1b) does notinvolve placing an aqueous solution of the diastereoisomericallyenriched complex of formula (II) in contact with a weak cationic resin.

In one particular embodiment, the steps 1a), when it is present, 1b and1c are performed in this order. This advantageous embodiment notablymakes it possible to minimize the amounts of resins used and thus theindustrial manufacturing cost.

-   -   Step 2)

Step 2) is directed towards isolating in solid form the purified complexof formula (II) obtained on conclusion of the combination of steps 1b)and 1c), and optionally also combined with step 1a).

This step of isolation in solid form may be performed according to anymethod that is well known to those skilled in the art, notably byatomization, by precipitation, by lyophilization or by centrifugation,advantageously by atomization.

In a preferred embodiment, step 2) comprises atomization.

Specifically, isolation in solid form of the purified complex of formula(II) by atomization makes it possible notably to dispense with the useof precipitation solvents.

The air inlet temperature in the atomizer is then typically between 150°C. and 180° C., notably between 160° C. and 175° C., advantageouslybetween 165° C. and 170° C. The outlet temperature is itself typicallybetween 90° C. and 120° C., preferably between 105° C. and 110° C.

Advantageously, the degree of purity of the purified complex of formula(II) diastereoisomerically enriched in the mixture of isomers II-RRR andII-SSS isolated on conclusion of step 2) is greater than 95%, notablygreater than 97%, preferentially greater than 97.5%, more preferentiallygreater than 98%, advantageously greater than 99%, said degree of puritybeing expressed as a mass percentage of the complex of formula (II)relative to the total mass obtained on conclusion of step 2).

The present invention also relates to the diastereoisomerically enrichedand purified complex of formula (II) which may be obtained according tothe purification process of the invention.

Preferably, the complex of formula (II) included in the compositionaccording to the invention described previously is thediastereoisomerically enriched and purified complex of formula (II)which may be obtained according to the purification process of theinvention.

EXAMPLES

The examples given below are presented as non-limiting illustrations ofthe invention.

Separation of the Groups of Isomers Iso1, iso2, iso3 and iso4 of theComplex of Formula (II) by UHPLC

A UHPLC machine constituted of a pumping system, an injector, achromatography column, a UV detector and a data station is used. Thechromatography column used is a UHPLC 150×2.1 mm−1.6 μm column (WatersCortecs® UPLC T3 column).

-   -   Mobile phase:    -   Route A: 100% acetonitrile and Route B: aqueous solution of        H₂SO₄ (96%) at 0.0005% v/v    -   Preparation of the test solutions:    -   Solution of the complex of formula (II) at 2 mg/mL in purified        water    -   Analytical conditions:

Column temperature 40° C. Sample temperature Room temperature (20-25°C.) Flow rate 0.3 ml/min Injection volume 1 μl UV detection 200 nmAnalysis time 20 min

-   -   Gradient:

Time % Acn % H₂SO₄ 0.0005% 0 1 99 3 5 95 12 10 90 15 25 75 16 1 99 20 199

Four main peaks are obtained. Peak 4 of the UHPLC plot, namely iso4,corresponds to a retention time of 6.3 minutes.

Preparation of the Butyl Hexaester of Formula (VI)

184 kg (570 mol) of dibutyl 2-bromoglutarate and 89 kg (644 mol) ofpotassium carbonate are mixed in a reactor and heated to 55-60° C. Anaqueous solution of 29.4 kg (143 mol) of pyclene in 24 kg of water isadded to the preceding preparation. The reaction mixture is maintainedat 55-60° C. and then refluxed for about 10 hours. After reaction, themedium is cooled, diluted with 155 kg of toluene and then washed with300 litres of water. The butyl hexaester is extracted into the aqueousphase with 175 kg (1340 mol) of phosphoric acid (75%). It is then washedthree times with 150 kg of toluene. The butyl hexaester is re-extractedinto a toluene phase by dilution with 145 kg of toluene and 165 kg ofwater, followed by basification with 30% sodium hydroxide (m/m) to reacha pH of 5-5.5. The lower aqueous phase is removed. The butyl hexaesteris obtained by concentrating to dryness under vacuum at 60° C., in ayield of about 85%.

Preparation of the Hexaacid of Formula (Ill)

113 kg (121 mol) of butyl hexaester are placed in a reactor along with 8kg of ethanol. The medium is brought to 55±5° C. and 161 kg (1207.5 mol)of 30% sodium hydroxide (m/m) are then added over 3 hours. The reactionmixture is maintained at this temperature for about 20 hours. Thebutanol is then removed by decantation of the reaction medium. Thehexaacid of formula (III) obtained in sodium salt form is diluted withwater to obtain an aqueous solution of about 10% (m/m). This solution istreated on an acidic cationic resin. The hexaacid of formula (III) inaqueous solution is obtained in a yield of about 90% and a purity of95%.

Preparation of the Hexaacid Gadolinium Complex of Formula (I)

-   -   Experimental protocol    -   Complexation and isomerization        -   Without acetic acid

418 kg (117 kg of pure hexaacid of formula (III)/196 mol) of an aqueoussolution of hexaacid of formula (III) at 28% by weight are placed in areactor. The pH of the solution is adjusted to 2.7 by addinghydrochloric acid, and 37 kg (103.2 mol) of gadolinium oxide are thenadded. The reaction medium is heated at 100-102° C. for 48 hours toachieve the expected isomeric distribution of the hexaacid of formula(III).

-   -   With acetic acid

Gadolinium oxide (0.525 molar eq.) is suspended in a solution ofhexaacid of formula (III) at 28.1% by mass.

99-100% acetic acid (50% by mass/pure hexaacid of formula (III)) ispoured into the medium at room temperature.

The medium is heated to reflux followed by distillation up to 113° C. bymass by refilling the medium with acetic acid gradually as the water isremoved. Once the temperature of 113° C. is reached, a sufficient amountof acetic acid to arrive at the starting volume is added.

The medium is maintained at 113° C. overnight.

-   -   Crystallization, recrystallization        -   Crystallization

The hexaacid gadolinium complex of formula (I) in solution is cooled to40° C., the primer is added and the agents are left in contact for atleast 2 hours. The product is then isolated by filtration at 40° C. andwashed with osmosed water.

-   -   Recrystallization

180 kg of the hexaacid gadolinium complex of formula (I) obtainedpreviously (solids content of about 72%) are suspended in 390 kg ofwater. The medium is heated to 100° C. to dissolve the product, and thencooled to 80° C. to be primed by adding a small amount of primer. Aftercooling to room temperature, the hexaacid gadolinium complex of formula(I) is isolated by filtration and drying.

-   -   Selective decomplexation

The dry product is placed in the reactor with osmosed water at 20° C.The mass of water added is equal to twice the theoretical mass ofhexaacid gadolinium complex of formula (I). 30.5% sodium hydroxide (m/m)(6.5 eq.) is poured into the medium at 20° C. At the end of the additionof NaOH, the medium is left in contact at 50° C. for 16 hours. Themedium is cooled to 25° C. and the product is filtered off on a bed ofClarcel.

-   -   Content of the mixture of diastereoisomers I-RRR and I-SSS

The ratio in which the various isomers of the complex of formula (I) arepresent in the mixture of diastereoisomers depends on the conditionsunder which the complexation and isomerization steps are performed, asis seen in Table 3 below.

TABLE 3 content of the mixture I-RRR and I-SSS as a function of thecomplexation/isomerization conditions Diastereoisomeric Content ofexcess in the hexaacid of mixture pH Temperature formula (III) TimeI-RRR and I-SSS 5.7  80° C. 40%  3 hours   19% 3.5  90° C. 50% 10 hours  49% 3.0 101° C. 40% 10 hours   68% 2.7 101° C. 28% 48 hours 98.04%

The additional steps of recrystallization and selective decomplexationmake it possible to increase the diastereoisomeric excess of the mixtureI-RRR and I-SSS (see Table 4).

TABLE 4 content of the mixture I-RRR and I-SSS after decomplexationcrystallization/recrystallization/selective After the first After Afterselective crystallization recrystallization decomplexationDiastereoisomeric 98.04% 99.12% 99.75% excess in the mixture I-RRR andI-SSS

Preparation of the Complex of Formula (II)

90 kg (119 mol) of the hexaacid complex of formula (I) and 650 kg ofmethanol are placed in a reactor. The mixture is cooled to about 0° C.and 111 kg (252 mol) of a methanolic solution of hydrochloric acid(8.25% of HCl in methanol) are then poured in while maintaining thetemperature at 0° C. The reaction medium is brought to room temperatureand stirring is then continued for 16 hours. After cooling to 0-5° C.,120 kg (1319 mol) of 3-amino-1,2-propanediol are added. The reactionmedium is then heated while distilling off the methanol under vacuumuntil a temperature of 60-65° C. is reached. The concentrate ismaintained for 16 hours at this temperature under vacuum. At the end ofcontact, the medium is diluted with 607 kg of water while cooling toroom temperature. The solution of the crude complex of formula (II) isneutralized with 20% hydrochloric acid (m/m). 978.6 kg of solution arethus obtained, with a concentration of 10.3%, representing 101 kg ofmaterial. The yield obtained is 86.5%, the purity of the complex offormula (II) is 92.3% (HPLC s/s). The amount of dicoupled impurities is6.4% (HPLC s/s).

Purification of the Complex of Formula (II)

-   -   Nanofiltration

The nanofiltration membrane used has a cut-off threshold of 200 daltons(Koch Membran System SR3D). This treatment is performed in the followingmanner:

The solution of crude complex of formula (II) is heated to 30° C. Thenanofilter is filled with said solution. The pump is switched on firstat a low rate to purge the system, then the rate of the nanofilter pumpis gradually increased to the desired recirculation rate (1.0 m³/h for amembrane of 2.5×40 inches). The system is then placed in totalrecirculation at 30° C. for at least 2 hours to establish a polarizationlayer. The medium is then passed to diafiltration at 30° C. under 2.5bar while keeping the volume constant by adding pure water until aconductivity of the retentate of less than 1000 μS is obtained. At theend of diafiltration, the medium is concentrated to obtain aconcentration of about 40% (m/m).

-   -   Treatment on resins

The solution of complex of formula (II) obtained from the nanofiltrationis diluted with purified water with stirring to obtain a 15% solution(m/m). This solution is eluted in series on 50 litres of strong anionicresins (FPA900) in OH⁻ form and then on 50 litres of weak cationicresins (HP336) in H⁺ form at a mean elution flow rate of 2V/V/H (2volumes of solution per volume of resin per hour). The resins are thenrinsed with about 450 litres of purified water until a refractive indexof less than 1.3335 is obtained.

The solution of complex of formula (II) is then concentrated by heatingto 50-60° C. under a vacuum of 20 mbar to reach a concentration of 35%(m/m).

-   -   Ultrafiltration

The ultrafiltration membrane is a UF 10KD Koch Spiral membrane.

The ultrafilter is fed with the preceding solution of complex of formula(II) at 35% heated to 40° C. The ultrafiltration is applied at a flowrate of 3 m³/h with a transmembrane pressure of 2.5-3 bar. The system isrinsed several times with 13 litres of apyrogenic purified water until afinal dilution of the complex of formula (II) of 25% (m/m) is reached.

-   -   Atomization

The complex of formula (II) is obtained in powder form by atomization ofthe preceding solution of complex of formula (II) concentrated to 25%.

The atomization is performed in the following manner:

The atomizer is equilibrated with apyrogenic pure water by setting theinlet temperature to 165° C-170° C. and adapting the feed rate such thatthe outlet temperature is between 105 and 110° C.

The concentrated solution of complex of formula (II) is then added andthe flow rate is adjusted so as to conserve the above parameters.

These operating conditions are maintained throughout the atomization,while ensuring good behaviour of the powder in the atomization chamberand at the atomizer outlet. It should notably be ensured that there isno adhesion of the product.

At the end of feeding the atomizer with the solution, the container ofthis complex of formula (II) and the atomizer are rinsed with apyrogenicpure water until maximum recovery of the powder is obtained.

A 99.6% pure complex of formula (II) is obtained.

This degree of purity was determined by reverse-phase liquidchromatography.

Composition According to the Invention and Results of Studies Thereon

-   -   Example of a manufacturing process in accordance with the        invention

The process for manufacturing a composition according to the inventionis performed according to the following steps:

a) 485.1 g (i.e. 0.5 M) of complex of formula (II) are dissolved inwater (qs 1 litre), heating the tank to a temperature of between 39 and48° C. and stirring the solution vigorously until this complex has fullydissolved in the water. The solution is then cooled to about 30° C.

b) 0.404 g (i.e. 0.2 mol/mol % relative to the proportion of complexadded in step a)) of DOTA (Simafex, France) is added with stirring tothe solution obtained in step a) via a solution of DOTA at 10% m/v.

c) Trometamol (Tris) is added to the solution obtained in step b) withstirring. The pH is then adjusted to a value of between 7.2 and 7.7 byaddition of hydrochloric acid solution with stirring.

d) The target concentration (0.5 mol/L) is obtained by adding water forinjection in two steps until a density value of between 1.198 and 1.219g/mL is obtained.

The liquid composition is then filtered through a polyethersulfonemembrane and placed in its final container, which is finally sterilizedat 121° C. for 15 minutes.

-   -   Example of a composition in accordance with the invention.

The following formulation is obtained by means of the process describedabove:

Ingredients Proportions in the composition Complex of formula (II) 485.1g (0.5M) DOTA** 0.404 g (1 mM, i.e. 0.2 mol/mol % versus complex) NaOHor HCl qs pH 7.2 to 7.7 Trometamol 1.211 g Free gadolinium* <1 ppm m/vWater for injection (injection-grade) qs 1 L *Measurement performed bythe colorimetric method with xylenol orange **expressed on an anhydrousand pure basis

-   -   Formulation tests performed

Various concentrations of trometamol from 0 to 100 mM were tested. Theresults of these tests showed that a content of 10 mM (0.12% w/v) wassufficient to ensure the pH stability of the formulation while limitingthe formation of degradation impurities.

Various concentrations of DOTA from 0 to 2.5 mM were tested. The resultsof these tests showed that a content of 1 mM, which corresponds to 0.04%m/v or 0.2 mol/mol %, makes it possible to ensure the absence of releaseof free Gd during the process and during the lifetime of the product.

-   -   Stability studies under accelerated conditions of a composition        according to the invention

The formulation of the preceding example is analysed just after itsmanufacture (T₀) and after storage at 40° C. for 6 months after itsmanufacture (T+6 months).

At T₀:

-   -   Purity evaluated by chromatography*: 99.6%    -   Concentration of Gd-DOTA: 0.007% (m/V)    -   Concentration of Gd: below 0.0001% (m/V)    -   pH: 7.5

At T+6 months:

-   -   Purity evaluated by chromatography*: 97.2%    -   Concentration of Gd-DOTA: 0.014% (m/V)−0.25 mM    -   Concentration of Gd: below 0.0001% (m/V)    -   pH: 7.5 * reverse-phase liquid chromatography

These results demonstrate that this formulation has good stability overtime.

-   -   Comparative stability studies

The stability of the compositions below was evaluated over time. Theterm “non-optimized AP” denotes the active principle, namely the complexof formula (II), obtained according to the process described in EP 1 931673. The term “optimized AP” denotes the diastereoisomerically enrichedand purified complex of formula (II) obtained via the process accordingto the invention.

AP [DOTA] Trometamol (0.5M) mol/mol % mM pH_(adjustment) C1 Not 0.3 —5.0 optimized C2 Optimized 0.2 — 7.5 C3 Optimized 0.1 — 7.5 C4 Optimized0.2 10 7.5 C5 Optimized 0.1 10 7.5 C6 Optimized 0.2 — 5.0 C7 Optimized0.1 — 5.0 Free Gd in ppm m/v DOTA-Gd in mol/mol % (xylenol) (LC formate)T 6 months T 6 months T 0 40° C. T 0 40° C. C1 <DL 0.18 0.27 0.3  C2 <DL<DL 0.02 0.05 C3 <DL <DL 0.02 0.05 C4 <DL <DL 0.02 0.05 C5 <DL <DL 0.020.08 C6 <DL <DL 0.03 0.03 C7 <DL <DL 0.02 0.07 * LC formate:chromatographic method involving fluorimetric detection. The separationis performed on a reverse-phase C18 grafted chromatography column withelution in gradient mode.

The results reported above indicate that formulation of thenon-optimized AP with free DOTA is not possible. The reason for this isthat the chelation excipient is entirely consumed by the trans-ligationreaction between the complex of formula (II) and DOTA and consequentlycan no longer play its role of trapping the leached Gd³⁺.

On the other hand, the diastereoisomerically enriched and purifiedcomplex of formula (II) obtained via the process according to theinvention may be formulated with free DOTA. Specifically, the absence offree Gd in the composition at 6 months, 40° C., is observed, this beingthe case irrespective of the pH of the formulation and whether or notbuffering species are present. In addition, the consumption of chelationexcipient is very low, since it does not exceed 0.08 mol/mol %.

1-15. (canceled)
 16. A process for purifying the complex of formula (II)below:

having a diastereoisomeric excess of at least 80% of a mixture ofisomers II-RRR and II-SSS of formulae:

comprising: 1) purifying the complex of formula (II) by: 1b) passing thecomplex of formula (II) through an ion-exchange resin, and 1c)ultrafiltering the complex of formula (II), and 2) isolating thepurified complex of formula (II) in solid form.
 17. The process of claim16, wherein an aqueous solution of the complex of formula (II) in step1b) is contacted with a strong anionic resin.
 18. The process of claim17 wherein step 1b) further comprises contacting the aqueous solution ofthe complex of formula (II) with a weak cationic resin.
 19. The processof claim 18, wherein the purifying step further comprises nanofilteringthe complex of formula (II).
 20. The process of claim 16, whereinstep 1) is performed in the order of 1b) and 1c).
 21. The process ofclaim 16, wherein step 1c) uses a membrane with a cut-off threshold ofless than 25 kD.
 22. The process of claim 16, wherein the complex offormula (II) before the purification process has a diastereoisomericexcess of at least 85%.
 23. The process of claim 16, wherein the complexof formula (II) before the purification process has a diastereoisomericexcess of at least 90%.
 24. The process of claim 16, wherein the complexof formula (II) before the purification process has a diastereoisomericexcess of at least 94%.
 25. The process of claims 16, wherein step 1)further comprises: 1 a) nanofiltering the complex of formula (II). 26.The process of claim 25, wherein the step 1) is performed in the orderof 1a), 1b) and 1c).
 27. The process of claim 16, wherein step 2)comprises atomizing the purified complex of formula (II) obtained instep 1).
 28. The process of claim 25, wherein step 1a) uses a membranewith a cut-off threshold of less than 300 daltons.
 29. The process ofclaim 16, wherein the complex of formula (II) obtained in step 2) has adegree of purity of greater than 95% evaluated by chromatography. 30.The process of claim 16, wherein the complex of formula (II) obtained instep 2) has a degree of purity of greater than 98% evaluated bychromatography.
 31. The process of claim 25, wherein the complex offormula (II) obtained in step 2) has a degree of purity of greater than99% evaluated by chromatography.
 32. The process of claim 25, whereinthe complex of formula (II) after six months has a degree of purity ofgreater than 97% evaluated by chromatography.
 33. The process of claim16, wherein the process for purifying the complex of formula (II)comprises: 1 a) nanofiltering the complex of formula (II) at atemperature from 20° C. to 40° C.; 1b) passing the complex obtained instep 1a) through an anion-exchange resin and then through acation-exchange resin; 1c) ultrafiltering the complex obtained in step1b) to remove endotoxins; and 2) isolating the purified complex offormula (II) in solid form, wherein the process is performed in theorder of 1 a), 1b), 1c) and 2).
 34. A process for preparing the purifiedcomplex of formula (II) below:

having a diastereoisomeric excess of at least 80% of a mixture ofisomers II-RRR and II-SSS of formulae:

comprising: a) complexing a hexaacid of formula (III) below:

with gadolinium to obtain the hexaacid gadolinium complex of formula (I)below:

b) isomerizing by heating the hexaacid gadolinium complex of formula (I)in an aqueous solution at a pH from 2 to 4 to obtain adiastereoisomerically enriched complex having a diastereoisomeric excessof at least 80% of a mixture of isomers I-RRR and I-SSS of the hexaacidgadolinium complex of formula (I), of formulae:

c) reacting the diastereoisomerically enriched complex with3-amino-1,2-propanediol to form the complex of formula (II); d)purifying the complex of formula (II) obtained in step c) by: d1)passing the complex of formula (II) through an ion-exchange resin, andd2) ultrafiltering the complex of formula (II); and e) isolating thepurified complex of formula (II) in solid form.
 35. The process of claim34, wherein step b) comprises: b1) isomerizing by heating the hexaacidgadolinium complex of formula (I) in an aqueous solution at a pH of from2 to 4 to obtain the diastereoisomerically enriched complex, b2)isolating the diastereoisomerically enriched complex by crystallization,and b3) purifying by crystallization the isolated diastereoisomericallyenriched complex obtained in step b2).
 36. The process of claim 34,wherein step c) comprises: c1) reacting the diastereoisomericallyenriched complex obtained in step b) with an alcohol of formula R₁OH inthe presence of an acid to form a triester of formula (VIII) below,

in which R₁ represents a (C₁-C₆) alkyl group, and c2) aminolysing thetriester of formula (VIII) with 3-amino-1,2-propanediol in the presenceof an acid.
 37. The process of claim 36, wherein step c) comprises: c1)reacting the diastereoisomerically enriched complex obtained in step b)with methanol in the presence of an acid to form the methyl triester offormula (IV) below, and

c2) aminolysing the methyl triester of formula (IV) with3-amino-1,2-propanediol in methanol in the presence of an acid, whereinthe methanol is removed by vacuum distillation, until a temperature ofat least about 55° C. is reached, the reaction medium being maintainedat this temperature under vacuum for a time greater than 5 hours beforebeing cooled to room temperature and diluted with water, wherein thetriester of formula (IV) is not isolated between step c1) and step c2).38. The process of claim 34, wherein the hexaacid of formula (III) isobtained by alkylating the pyclene of formula (V):

with dibutyl 2-bromoglutarate to obtain the butyl hexaester of formula(VI):

and hydrolyzing the butyl hexaester of formula (VI) to obtain thehexaacid of formula (III).
 39. A composition comprising: 1) a complex offormula (II) below:

having a diastereoisomeric excess of at least 90% of a mixture ofisomers II-RRR and II-SSS of formulae:

and 2) a free macrocyclic ligand, wherein the composition has aconcentration of free gadolinium of less than 1 ppm (m/v).
 40. Thecomposition of claim 39, wherein the free macrocyclic ligand is1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
 41. Thecomposition of claim 39, wherein the composition comprises from 0.002 to0.4 mol/mol % of free macrocyclic ligand relative to the complex offormula (II).
 42. The composition of claim 39, wherein the complex offormula (II) has a diastereoisomeric excess of at least 94%.
 43. Thecomposition of claim 39, wherein the degree of purity of the complex offormula (II) is greater than 97% evaluated by chromatography.
 44. Amethod of performing magnetic resonance imaging of a subject,comprising: (a) administering to the subject a sufficient amount of thepurified complex of claim 1; and (b) imaging the subject using amagnetic resonance imaging device.
 45. The method of claim 44, whereinthe subject is a human.